Process for the preparation of creatinine amidohydrolase

ABSTRACT

CREATININE AMIDOHYDROLASE AND CREATINE AMIDINOHYDROLASE ARE PREPARED BY A PROCESS COMPRISING CULTURING (A) ALCALIGENCE SPEC. WS 51400 OR (B) PENICILLIUM WS 90001; IN A CREATININE-CONTAINING MEDIUM, DIGESTING SAME, OBTAINING THE CREATININE AMIDIODOHYDROLASE IN SUBSTANTIALLY PURE FORM FROM THE WATER-SOLUBLE DIGESTION FRACTION AND, OPTIONALLY SEPARATING BY ION EXCHANGE CHROMATOGRAPHY ANY CREATININE AMIDINOHYDROSALE PRESENT.

States Patent Oi 3,806,420 Patented Apr. 23, 1974 3,806,420 PROCESS FORTHE PREPARATION OF CREATININE AMIDOHYDROLASE Gunter Holz, JohannaGramsall, Michael Nelboeck- Hochstetter, and Hans Ulrich Bergmeyer,Tutzing, Upper Bavaria, Germany, assignors to Boehringer Mannheim GmbH,Manuheim-Waldhof, Germany No Drawing. Filed May 2, 1972, Ser. No.249,588 Claims priority, application Germany, May 5, 1971, P 21 22 294.6Int. Cl. C12d 13/10 US. Cl. 195-66 R 18 Claims ABSTRACT OF THEDISCLOSURE Creatinine amidohydrolase and creatine amidinohydrolase areprepared by a process comprising culturing (a) Alcaligenes spec. WS51400 or (b) Penicillium WS 90001; in a creatinine-containing medium,digesting same, obtaining the creatinine amidohydrolase in substantiallypure form from the water-soluble digestion fraction and, optionallyseparating by ion exchange chromatography any creatine amidinohydrolasepresent.

The invention is concerned with two new enzymes, creatinineamidohydrolase and creatine amidinohydrolase, and with a novel processfor the preparation thereof from microorganisms.

In clinical chemistry, especially for the functional diagnosis of thekidney, the determination of the intermediate and end products ofprotein metabolism play an important part. The products of thismetabolism include creatinine and creatine. Methods for thedetermination of creatinine have already been frequently described. Mostof the known methods are based on the nonenzymatic Jaife reaction which,however, sufiers from the disadvantage of being non-specific.

Furthermore, there has also been described a specific microbiologicalmethod of determination of creatinine With the use of isolated bacteriain which washed cell suspensions Were used for the measurement of thecreatinine and the formation of urea and ammonia was used as a measureof the enzyme action. However, it was not possible to obtain solubleenzyme extracts capable of breaking down creatinine. A prerequisite forthe provision of a specific creatinine determination with the help ofenzymes is, however, the discovery of appropriate soluble enzymes whichcan catalyze specific and measurable reactions of creatinine. With thisobject in view, experiments have been carried out with, for example,Corynebacterium, Psaudomonas aeroginosa, Pseudomonas ovalis, Pseudomonaseisenbergii and Clostridia, but all of these experiments wereunsuccessful.

Roche, Lacombe and Girard (BBA 6, 210/1950) characterized, in two typesof Psuedomonas, creatinase, creatininase and a glycocyaminase asspecific enzymes which liberate urea from their substrates from theguanidino group. Furthermore, Akamatsu et a1. (Enzymologia, 15, 122, 158173/1951) found, in soil bacteria, an enzyme which they calledcreatine-mutase and which brings about the equilibrium adjustmentbetween creatinine and creatine. The following course of breakdown ofthe creatinine was assumed:

Creatinineccreatineeurea and sarcosine glycine NH We have now found andisolated, in a state of high purity, two enzymes in microorganisms,which enzymes appear to decisively participate in the breakdown ofcreatinine, viz., a creatinine amidohydrolase and creatineamidinohydrolase. The present invention is based on the discovery thattwo microorganisms are particularly eflicacious for providing theenzymes creatinine amidohydrolase and creatine amidinohydrolase.

Thus, this invention comprises a process for preparing said enzymes. Theprocess comprises, essentially, culturing a microorganism when themicroorganism is (a) Alcaligenes spec. WS 51400 or (b) Penicillium WS90001; in a creatinine-containing medium, digesting same and obtainingthe creatinine amidohydrolase in pure form from the water-solubledigestion fraction by known biochemical purification and fractionationmethods carried out at a pH value above 7.0, with the use of a test inwhich creatine formed from added creatinine is determined in knownmanner. Thereafter, if desired, any creatine amidinohy drolase presentis separated by exchange chromatography from the creatinineamidohydrolase obtained.

The following reactions are catalyzed by two enzymes provided by thepresent invention:

(a) creatinine amidohydrolase creatine amidlnohydrolase Creatine 11 0sarcoslne urea Since Equation (a) is that of an equilibrium reactionwith the taking up or removal of water, the enzyme which participates inthis reaction is called creatinine amidohydrolase.

As starting material for obtaining the two new enzymes, there can, ingeneral, be used said microorganisms in which the desired enzymes areadaptively enriched, this usually taking place by culturing themicrooganisms in the presence of or with the addition of creatinine.

A water-soluble fraction is obtained by digestion of the microorganismso obtained. The usual digestion methods can be used, depending upon thestrength or resistance of the cell membrane of the microorganism used.High pres sure dispersion and ultrasonic digestion have proved to beespecially useful. Further examples of digestion methods include the useof distintegration mills, for example those of Balutini and Schlossmann,and digestion methods working on a similar basis. Chemical or enzymaticdigestion methods can also be used.

The creatinine amidohydrolase can be enriched from the so obtainedwater-soluble fraction by known biochemical purification methods byutilizing its ability to convert creatinine into creatine for testingthe result of any biochemical purification method used. The creatineformed can be determined by known methods, for example, by the additionof adenosine triphosphate and measurement in the usual way of theadenosine diphosphate formed. It is, however, necessary to operate at pHvalues above 7 and to select a purification method which can be used atthese pH values.

According to a further method of following the progress of theenrichment process, the creatine formed is split with creatineamidinohydrolase, with the formation of sarcosine and urea, and then todetermine the urea in the usual way, for example, by the use of urease.

The extent to which the purification is carried out depends upon theintended use of the enzyme or enzymes. If it is desired to obtain apreparation suitable for the enzymatic determination of creatinine, asingle purification step sufiicies. Examples of such purification stepsinclude a polyanion treatment, fractionation with organic solvents orchromatography. With the use of the abovementioned methods ofdetermination, it is possible, in a simple manner to ascertain thecontent of the desired enzyme and to enrich it to a considerable extentin a single step.

Since a comparatively long storage of the collected and frozenmicroorganisms could lead to a decrease of activity of the desiredenzymes, the cells are preferably further worked up as quickly aspossible after collection. Two microorganisms used for carrying out theprocess of the present invention are Alcaligenes spec. WS 51400 of theAchromo bactericeae and Penicillium WS 90001. These two microorganismsare deposited, under the stated numbers, in the collection of theBacteriological Institute of the Technical University, Munich, Germany,at Weihenstephan.

Alcaligenes spec. WS 51400 possesses the following properties: strictlyoxidative gram-negative, short rodlets with peritrichous flagellation.The microorganism is weakly oxidase-positive, alkalizes litmus milk andis capable of denitrification. Furthermore, the following propertieshave been ascertained:

Growth at 4 C Growth at 41 C. Gelatine liquifaction a Tributyrin fissionEgg yolk reaction Pigment formation Fermentation of:

According to present knowledge, this microorganism belongs to theAchromobacteraceae family and is to be assigned, with great probability,to the genus Alcaligenes.

The other preferred microorganism, namely WS 90001, is a fungus of thePenicillium genus.

In order adaptatively to enrich the desired enzymes in themicroorganisms to be used according to the present invention, these arepreferably cultured with the addition of creatinine to the nutrientmedium. The microorganisms are advantageously allowed to grow on anutrient substrate which contains glucose or glycerol, as a source ofcarbon and creatinine, as well as salts and vitamins in the amounts andcompositions known in microbiology. An especially preferred nutrientmedium has the following composition:

0.5 wt. percent glucose or glycerol 0.5 wt. percent creatinine 0.08 wt.percent ammonium sulphate 0.02 wt. percent magnesium sulphate hydrate0.05 wt. percent yeast extract 1 mg. nicontinic acid 1 mg.thiamine-p-aminobenzoic acid 1 mg. vitamin B 0.1 mg. biotin togetherwith traces of iron sulphate, calcium chloride and manganese sulphate,dissolved in M/ potassium phosphate buffer with a pH of 6 in the case ofthe Penicillium or with a pH of 7 in the case of Alcaligenes.

A strain of the mircoorganisms to be used according to the presentinvention is obtained in the usual way on agar tilted tubes by theaddition of 2% agar to a suitable nutrient substrate, preferably to theabove-described nutrient substrate, to which 5 m1./ liter of a 0.05%bromothymol blue solution is also added. Under optimum growthconditions, the indicator changes color, in the case of the Penicilliumafter 4 to 6 days and in the case of the Alcaligenes in 2 to 3 days. Thecolor change is clearly into the alkaline region, brought about by thebreakdown of creatinine and creatine. Creatinine-utilizingmicroorganisms which do not bring about the breakdown according to theabove-mentioned metabolic scheme with the abovementioned enzymes, do notbring about this alkalization.

The microorganisms to be used according to the present invention permitthe economic production of creatinine amidohydrolase and creatineamidinohydrolase from the water-soluble protein fractions obtained froma digest of the microorganisms.

Since creatinine amidohydrolase and also creatine amidinohydrolasequickly lose their activity at pH values of 6 and below and exhibit thegreatest stability at pH values of about 8.0, the digestion ispreferably carried out with the use of an alkaline buffer, 0.1 Mpotassium phosphate buffer (pH 8.0) being preferably used. The bufferand the buffer concentration used should preferably be such that anyfurther enrichment of the enzymes can also be carried out in the bufferused for the digestion. When carrying out the digestion by high pressuredispersion, usually at about 700 to 800 ats., further purification can,when using a polyanion treatment, he carried out without previousseparation of the cell residues. Examples of polyanions which can beused include protamine sulphate and water-soluble polyethyleneimines. Itis preferred to add a water-soluble polyethyleneimine, for example, inthe form of a 10% solution of pH 8. When using a solution of thisconcentration, an about 5% addition thereof, referred to the digestvolume, is necessary to achieve complete precipitation. The precipitatecan then be separated by physical means, for example, by filtering orcentrifuging, the desired enzymes remaining in the supernatant.

Instead of a polyanion precipitation, a precipitation with organicsolvents can also be advantageous, isopropanol preferably being used. Inthis case, the cell residues are first removed after the digestion andthen the organic solvent is added at ambient temperature. Theisopropanol fractionation is preferably so carried out that at about 25C., 800 ml. of isopropanol is added per litre of digest solution and theprecipitate separated off. The supernatant obtained is again mixed with500 ml. isopropanol per litre and the precipitate, which contains thetwo desired enzymes, separated Off.

A combined use of the two above-mentioned methods, namely polyanionprecipitation and subsequent fractionation with organic solvents, isespecially preferred.

Not only the products obtained with a single enrichment step but alsothose with the two combined steps are sufficiently pure for use in aspecific creatinine determination. The enzymes creatinine amidohydrolaseand creatine amidinohydrolase are hereby always obtained in a mixture.If it is desired to separate the enzymes, the preparation so obtained issubsequently separated by exchange chromatography, a weakly basic ionexchanger, such as diethylaminoethyl-Sephadex ordiethylaminoethylcellulose having proved to be especially useful.

The enzymes are adsorbed on the exchanger, for example ondiethylaminoethyl-"Sephadex, at a low ion concentration, preferablybelow 0.1 M and more preferably of about 0.01 to 0.05 M. Subsequently,the exchanger is washed with an ion concentration of about 0.1 M,nonactive accompanying proteins thereby being removed. Creatinineamidohydrolase can then be eluted with 0.2 M buffer solution, whereasthe creatine amidinohydrolase still remains on the exchanger. It canalso be eluted by increasing the ion concentration to 0.5 M, forexample, by using 0.2 M potassium phosphate bufler (pH 8.0) with acontent of 0.3 M sodium chloride or potassium chloride or a similarsalt.

The above-described preferred embodiment of the process according to thepresent invention, which combines an isopropanol fractionation with anexchange chromatography, leads to an approximately 100 to 150 foldenrichment of the enzymes and gives a creatinine amidohydrolasepreparation with a specific activity of more than 200 u./mg. Thecreatine amidinohydrolase is hereby enriched about 100 fold, apreparation with an activity of 3 u./rng. being obtained.

According to another embodiment of the process according to the presentinvention, a very simple high purification of the desired enzymes can beobtained without separation when, in a batch process, sufiicientexchanger is added to adsorb both enzymes. The exchanger is thenseparated and thereafter washed and subsequently eluted in the mannerdescribed above.

A further possibility for the purification and enrichment of the twoenzymes is by means of salt precipitation or salt fractionation, forexample, with the use of ammonium sulphate. Thus, when using ammoniumsulphate, creatinine amidohydrolase precipitates at a concentration of2.2 M and creatine amidinohydrolase or the mixture of the two enzymes ata concentration of 2.7 M.

Salt precipitation can also be used for obtaining the enzymes fromsolutions thereof, such as are obtained, for example, by elution of theexchanger. By dialysis at about 4 C., expediently against dilutedbuffer, for example 0.02 M diethanolamine buffer (pH 8.0), the enzymescan be freed from salts and other low molecular weight accompanyingmaterials.

The preparations so obtained can be stored for several months in afrozen state, without loss of activity.

If only creatinine amidohydrolase is desired, a further purification andenrichment step can also comprise a heating step at 60 C. Such heatingfor a period of 5 minutes does not lead to any noticeable reduction ofthe creatinine amidohydrolase activity. However, in contradistinctionthereto, the creatine amidinohydrolase loses its activity under theseconditions.

The new enzymes according to the present invention can be used forsicientific purposes, as well as for the specific determination ofcreatinine and creatine.

The following examples are given for the purpose of illustrating thepresent invention.

EXAMPLE 1 Enrichment of active fungal mycelia in a submersed shakeculture 2 litres of a nutrient substrate containing, by weight, 1%glucose, 0.5% creatinine, 0.08% ammonium sulphate, 0.02% magnesiumsulphate heptahydrate, 0.05% yeast extract, 1 mg. each of nicotinicacid, thiamine-p-aminobenzoic acid and vitamin B 0.1 mg. biotin andtraces of iron sulphate, calcium chloride and manganese sulphate, in0.10 M potassium phosphate buffer (pH 6) are inoculated in a shakingflank with 200 ml. of a 48 hours old pre-culture of Penicillium WS 90001and cultured in a shaking apparatus for 4 days. The creatinine contentdrops in this time from 0.5% weight to scarcely 0.1% by weight and,after this time, the glucose is almost used up. Furthermore, the pHvalue increases to 7.5 to 8.0. There are thus obtained 3 to 4 g.Penicillium WS 90001 dry weight per litre of culture solution with acontent of 45 to 60 IU/ g. dry weight of creatinine amidohydrolase.

EXAMPLE 2 In a culture flask containing 15 litres of the nutrient mediumdescribed in Example 1 are cultured, with vigorous aeration, 1.5 litresof a well grown pre-culture of Alcaligenes spec. WS 51400. During thewhole culturing period, the pH is maintained constant at 7.0. Thecreatinine and the glucose content are followed continuously. Thecreatine utilization take place mainly in the second third of the logphase, whereas the glucose is broken down first. The culture ismaintained at 30 C. After 35 hours, 1.5 -g. dry bacteria mass can becollected per litre of culture solution. The specific activity ofcreatinine amidohydrolase is 1600 IU/ g. dry weight.

EXAMPLE 3 Alcaligenes spec. WS 51400. During the whole culturing Example2, in a working volume of 60 litres. As soon as the desired stage ofgrowth is reached, fresh nutrient substrate is added to the culturevessel and grown culture solution is removed from the culture vessel atthe same rate. The dilution rate (flow velocity/working volume) isthereby 0.13 to 0.18 and preferably about 0.16. 500 litres of air arepassed through the culture solution per hour. There is thus obtained, bycontinuous culturing, a yield of 3 to 5 g. dry bacteria mass per litre,with a specific activity such as is obtained by the batch proceessdescribed in Example 2.

Alcaligenes spec. WS 51400 is thus well suited for continuous culturingin the manner described above.

EXAMPLE 4 Isolation of highly purified creatinine amidohydrolase Thecells -(1 kg. dry weight) are separated from a culture batch ofAlcaligenes spec. WS 51400 cultured in the presence of creatinine, thenmade up with 0.1 M potassium phosphate butler (pH 8.0) to 20 litres and,without cooling, digested by high pressure dispersion at a pressure ofabout 800 ats. The extract is mixed with 5 vol. percent of a 10%polyethyleneimine solution (mol. weight about 1800) of pH 8.0 (about 1litre). After the addition of potassium chloride (0.01 M end molarity)and ammonium chloride (0.1 M end molarity), there are added, within thecourse of 10 minutes, 0.8 volume of aqueous isopropanol per litre ofextract, followed by stirring for 30 minutes at ambient temperature.

The copious precipitate obtained is centrifuged 01f, whereafter 0.45volume of 90% isopropanol are added per litre of supernatant.

The precipitate so formed, which contains creatinine amidohydrolase andcreatine amidinohydrolase, in separated off and taken up in 400 ml. 0.02M potassium phosphate buffer (pH 8.0). Undissolved residue is separatedoff and the supernatant is applied to a diethylaminoethyl- Sephadexcolumn (3.5 cm. x 1 m.) which has been equilibrated with the samebulfer. Thereafter, the column is washed with 1 vol. 0.1 M potassiumphosphate buffer (pH 8.0) and then eluted with 0.2 M potassium phosphatebuffer (pH 8.0). The creatinine amidohydrolase-containing fractions arecombined and adjusted at pH 8.0 with ammonium sulphate to 2.2 M. Theprecipitated enzyme is centrifuged off and dissolved in 0.02 Mdiethanolamine buffer (pH 8.0) to give a volume of ml. and then dialyzedfor 4 hours at 4 C. against the same butter. There is thus obtained atotal yield of 64% of a preparation with a specific activity of 303u./mg. The following Table I summarizes the activities and yieldsobtained in the individual steps of this process.

When carrying out the digestion ultrasonically, instead of by highpressure dispersion, the content of creatinine amidohydrolase, withabout the same specific activity, can be increased 2.5 fold, referred tothe dry weight of the microorganisms used.

The creatinine amidohydrolase obtained as described above has anequilibrium constant Creatine creatinine The Michealis constant forcreatinine as substrate M 37 C.;pH 8.0).

KM=3.3 10 37 c.; pH 8.0).

EXAMPLE Separation of creatinine amidohydrolase and creatineamidinohydrolase The procedure described in Example 4 is repeated.However, after elution of the creatinine amidohydrolase from theexchanger column, the creatine amidinohydrolase is eluted with 0.2 Mpotassium phosphate buffer (pH 8.0), containing 0.3 M sodium chloride.The following Table II shows the details.

TABLE II [Separation of creatinine amidohydrolase and creatineamidinohydrolase The procedure described in Example 4 is repeated.However, the diethylaminoethyl-Sephadex chromatography is replaced by adiethylaminoethyl Sephadex batch process.

Starting from 100 g. Alcaligenes spec. WS 51400, 100 ml. of the secondisopropanol precipitate are dissolved in 0.02 M potassium phosphatebuffer (pH 8.0) and mixed with such an amount of diethylaminoethyl-Sephadex that in the supernatant there remain about 5% of each of theenzymes (about 20 g. of moist, pressed out exchanger). The exchanger isfiltered off, washed with about 100 ml. 0.08 M potassium phosphatebuifer (pH 8.0) and, for the joint elution of the two enzymes, stirredwith 100ml. 0.2 M potassium phosphate buffer (pH 8.0), containing 0.3 Mpotassium chloride, for 15 minutes at 4 C. and subsequently filtered.Both enzymes are present in the eluate. The following Table III showsthat, by precipitation with ammonium sulphate, there can be obtained apreparation containing 31.5 u./mg. creatinine amidohydrolase and 1.1u./mg. creatine amidinohydrolase.

TABLE III [Isolation of an enriched enzyme mixture of creatinineamidohydrolase and creatine amidinohydrolase from 100 g. (dry weight)Alcaligenes spec. WS 51400] 8 EXAMPLE 7 7 g. (dry weight) Alcaligenesspec. WS 51400, grown in the presence of creatinine, were collected anddigested ultrasonically at pH 8.0. The suspension obtained was mixed, asdescribed in Example 1, with 0.8 volume isopropanol, stirred for 30minutes at ambient temperature and centrifuged. The supernatant is mixed'with a further 0.45 volumes isopropanol and again centrifuged. Theprecipitate is taken up as described in Example 1 and chromatographedover a diethylaminoethyl-Sephadex column, only the creatinineamidohydrolase thereby being eluted. The following Table IV shows thedetails of this process. The preparation obtained was only suitable forthe determination of creatinine.

TABLE IV U. Protein Yield, Step (25 C.) in g. U./mg. percent Ultrasonicdigest. 1.5X10 1.780 0.85 100 Second isopropanol addition(precipitate) 1. 2X10 0. 119 10. 1 Diethylaminoethyl- Sepha dex eluate0. 7 X10 0.028 25 47 In the experiments described in the above examples,the creatine formed from creatinine in the presence of creatinineamidohydrolase was determined by the addition of adenosine triphosphate(ATP); in the presence of creatine phosphokinase ('CPK), the creatine isconverted into creatine phosphate and adenosine diphosphate (ADP). ADPformed is converted into ATP with pyruvate kinase (PK) and lactatedehydrogenase (LDH), as Well as phosphoenol pyruvate (PEP), with theconsumption of NADH as measured optically. These known steps can beillustrated as follows:

OPK (1) creatine ATP creatine phosphate ADP PK (2) ADP PEPpyruvate-l-ATP LDH (3) pyruvate NADH+ H+ lactate NAD+ (b) Penicillium WS900001; in a creatinine-containing medium, digesting said microorganismsand recovering creatinine amidohydrolase from the water-solubledigestion fraction in substantially purified form by known biochemicalpurification methods carried out at a pH of about 7.0.

2. Process as claimed in claim 1 wherein said creatinine amidohydrolaseobtained in substantially purified form is subjected to exchangechromatography to separate therefrom any creatine amidinohydrolase.

3. Process as claimed in claim 1 wherein said digesting is carried outmechanically.

4. Process as claimed in claim 3 wherein the mechanical digesting iscarried out by means of high pressure dispersion or by use of adigestion mill or ultrasonically.

5. Process as claimed in claim 4 wherein said digesting is effected byhigh pressure dispersion carried out at a pressure of from 700 to 800atmospheres.

6. Process as claimed in claim 1 wherein said digesting is carried outat a pH value of about 8.0.

7. Process as claimed in claim 1 wherein said watersoluble digestionfraction is purified by a polyanion fractionation.

8. Process as claimed in claim 7 wherein the polyanion fractionation iscarried out by a polyethyleneimine precipitation at a bufierconcentration of about 0.1 M.

9. Process as claimed in claim 1 wherein a purification is carried outby means of an isopropanol fractionation in one or more steps.

10. Process as claimed in claim 9 wherein the isopropanol fractionationis carried out with 90% isopropanol at about 25 C.

11. Process as claimed in claim 2 wherein the enzymes are adsorbed on aweakly basic ion exchanger at an ion concentration below 0.1 M, theexchanger is washed with about 0.1 M butter and creatinineamidohydrolase and creatine amidinohydrolase are eluated with an ionconcentration of 0.2 M and 0.5 M, respectively.

12. Process as claimed in claim 11 wherein the enzymes are adsorbed onthe weakly basic ion exchanger at an ion concentration of 0.01 to 0.05M.

13. Process as claimed in claim 1 wherein the enzymes are isolated fromsolutions thereof by a salt precipitation 10 or salt fractionation,creatinine amidohydrolase precipitating at a concentration of 2.2 M andcreatine amidinohydrolase or a mixture of the two enzymes precipitatingat a concentration of 2.7 M.

14. Process as claimed in claim 13 wherein the solutions used for thesalt precipitation or salt fractionation are first purified by dialysis.

15. Process as claimed in claim 14 wherein the dialysis carried out atabout 40 C. against a diluted buffer.

16. Process as claimed in claim 15 wherein the diluted buffer is 0.02 Mdiethalolamine buffer with a pH of 8.0.

17. Process as claimed in claim 1 wherein said creatinine-containingmedium also contains glucoses or glycerine.

18. Process as claimed in claim 17 additionally containing salts andvitamins.

References Cited Koppel: Archives of Biochemistry, vol. 19, pp. 171-172(1949).

Roche et al.: Biochimica et Biophysica Acta, vol. 6, pp. 210-216 (1950).

LIONEL M. SHAPIRO, Primary Examiner

